The pseudouridine (psi) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles. Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M. musculus, H. sapiens and Trypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 psi residues. The number of psi residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA of D. melanogaster and M. musculus. Despite this, all of the psi residues were found in three domains, II, IV and V. All three are at or have been linked to the peptidyl transferase center according to the literature. Comparison of the sites for psi among the species examined revealed four conserved or semi-conserved segments. One is the region 1911 to 1917, which contains three psi or modified psi in almost all species examined. This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition. Three additional sites were at the peptidyl transferase center itself. The juxtaposition of the conserved sites for psi with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for psi in ribosome function. We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence of Z. mays chloroplasts and D. melanogaster LSU RNA, correlate the secondary structural context for all known psi residues in ribosomal RNA, and compare the sites for psi with those known for methylated nucleosides in H. sapiens.